A Novel Approach For Cryopreservation Of Adipose-Derived Mesenchymal Stem Cells And Their Constructs

Cryopreservation is an effective and indispensable method for the long-term storage of cells,tissues,organs and even recently emerging biological constructs,which is widely used in the field of biomedicine such as stem cell therapy,bio-tissue engineering and regenerative medicine.Usually,the cryopreservation includes slow freezing?two-step?cryopreservation and vitrification.Currently,the slow freezing is the most widely used,but it has still some shortcomings,especially the cryopreservation of stem cells and biological materials.During slow freezing,ice crystals form in cells or biomaterials,which can cause mechanical damage to cells and biological materials,as well as solute damage.At the same time,there may be effects on stem cell function during the process of slow freezing.Vitrification cryopreservation can avoid ice crystals formation during cooling,but traditional vitrification uses high concentrations of cryo-protectant agents?CPAs?inevitably,which can cause cytotoxic damage.Therefore,the search for low concentrations of CPAs vitrification cryopreservation is very necessary.However,the greatest challenge in the low-CPAs vitrification is recrystallization during rcwarming.Therefore,understanding the causes of recrystallization and the influencing factors is very important.In this paper,we studied the probability of intracellular ice formation and recrystallization and the cell viability after freeze-thawing under the different cooling rates and in different solutions when the cells were in both adherent and suspended stat.The results showed the probability of intracellular ice and recrystallization increased that with the increase of the cooling rate.At the same time,the formation of intracellular ice and recrystallization in the presence of 1 M DMSO was delayed than in PBS.Although the intracellular ice and recrystallization are more likely to be formed in 1 M DMSO solution,the survival rate post-rewarming is higher than in PBS solution,because ice formation is only one of the causes of cell damage,and low CPAs can protect cells during freezing-thawing,such as stabilizing cell membrane.Relative to the suspension cells,the probability of intracellular ice formation of adherent cells is much higher during the same freezing-thawing,but the cell viability is higher than the suspension cells post-rewarming,because the cell gap junctions can avoid excessive dehydration of cells and protect cells during the freezing-thawing process.It has been reported that nanoparticles can promote ice crystals formation.Fe₃O₄ nanoparticles have been utilized in cryopreservation of tissue,but the effect on the cryopreservation solution has not yet been studied.Therefore,we observed the effect of different concentrations of Fe₃O₄ nanoparticles on ice crystal formation by cryogenic microscopy.The results showed that when Fe₃O₄ nanoparticles were added into the solution,the temperature of ice nucleation increases about 10 ?,and the growth rate of ice crystals was also increase.This is because the nanoparticles can be considered as nucleating agents for ice crystals formation and promote the nucleation and growth of ice crystals.Alginate hydrogels are commonly used to inhibit devitrification in the process of rewarming in the previous study of low-CPAs vitrification,but microscopy and research of alginate are less.We used cryogenic microscopy to study the effect of alginate hydrogel with 200?m thickness on ice crystal formation and the effect of cryogenic conditions on the structure of alginate hydrogel microcapsules.We found that alginate hydrogels had only a small amount of ice crystals formed,and no recrystallization occurred during rewarming.This indicates that alginate hydrogel,to some extent,can inhibit ice crystals formation and recrystallization.At the same time,we found that the morphology of the alginate hydrogel microcapsules is not affected by freeze-thaw cycle when the CPA concentration is greater than 1 M.In the presence of a lower concentration of penetrating CPAs?2 M?,the boundaries of ice crystals are often sharp,whereas in the case of high concentration of penetrating CPAs?4 M?or at low penetrating CPAs of 2 M and 1.3 M trehalose,the boundaries of ice crystals are mostly rounded and smooth.Therefore,the addition of trehalose will greatly improve the conservation efficiency during cryopreservation of cells and other biological samples.We used DSC method to study the effect of alginate hydrogel microspheres on the recrystallization with different concentrations.The results showed that there are much less ice crystals in low CPAs with alginate hydrogel microspheres than without hydrogel microspheres,indicating that the hydrogel hydrogel can promote water to the vitrification state.In order to solve the problem of recrystallization during the low-CPA vitrification,we have developed a new method that combines the Fe₃O₄ nanoparticles with the microencapsulated alginate hydrogel to successfully realize the low-CPA vitrification cryopreservation of the stem cell-alginate hydrogel constructs.In the past,most of the single hydrogel were used in the process of cell encapsulation.In our experiments,we used hydrogel microcapsules with "core-shell" structure.The outer shell not only protected the cells from solution shear damage during encapsulation,but also avoided cells from the ice damage during rewarming,meanwhile,the shell can also isolate Fe₃O₄ nanoparticles and cells.In the rewarming process,alginate hydrogels can inhibt the occurrence of recrystallization to a certain extent.Meanwhile,under the alternating magnetic field,the Fe₃O₄ nanoparticles can generate uniform heat to further suppress the local and the overall solution recrystallization or devitrification occurs.The combination of both methods can improve the real-time survival rate of stem cells,maintain the biofunction of stem cells and improve greatly the long-term survival rate of stem cells?eg,adherent efficiency?.Compared with traditional rewarming,the nano-warming increases the adherent efficiency of stem cells from 24%to 68%,which is especially important for its later application.In addition,the nano-warming could maintian the integrity of the stem cell-construct structure and cell efficient proliferation in 3D culture.This new nano-warming method has potential value for promoting the application of stem cells and their-constructs to clinical field.Moreover,in order to exclude the influence of osmotic pressure of the solution on the experimental results and perform a more comprehensive detection of cell activity during biological experiments,we developed a novel method for dynamic detection of solution osmotic pressure and cells activity by the combination of microcantilever and cells.Compared with the general method of cell activity detection,our method can also be used for label-free anti-cancer drugs screening and exhibit better sensitivity.